Antisense oligonucleotides of human regulatory subunit RI.sub.α of cAMP-dependent protein kinases

ABSTRACT

Antisense oligonucleotides of human regulatory subunit RI-alpha of cAMP-dependent protein kinases are disclosed along with pharmaceutical compositions containing these oligonucleotides as the active ingredients. These antisense oligonucleotides have been shown to inhibit the growth of several cancer cell lines including HL-60, human colon carcinoma LS-174T, neuroblastoma cells, breast cancer cells, and gastric carcinoma cells. In addition, these oligonucleotides can inhibit the growth of human colon carcinoma cells transplanted in athymic mice.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of U.S. applicationSer. No. 07/680,198, filed Apr. 5, 1991, now abandoned, is acontinuation-in-part of U.S. application Ser. No. 07/607,113, filed Nov.2, 1990, now abandoned the contents of which are fully incorporated byreference herein.

FIELD OF THE INVENTION

The invention is in the field of medicinal chemistry. In particular, theinvention relates to certain antisense oligonucleotides and the usethereof for the treatment of cancer.

BACKGROUND OF THE INVENTION

The present invention was made with government support. Accordingly, theUnited States government has certain rights in the invention.

Control mechanisms for cell growth and differentiation are disrupted inneoplastic cells (Potter, V.R. (1988) Adv. Oncol. 4, 1-8; Strife, A. &Clarkson, B. (1988) Semin. Hematol. 25, 1-19; Sachs, L. (1987) CancerRes. 47, 1981-1986). cAMP, an intracellular regulatory agent, has beenconsidered to have a role in the control of cell proliferation anddifferentiation (Pastan, I., Johnson, G. S. & Anderson, W. B. (1975)Ann, Rev. Biochem. 44, 491-522; Prasad, K. N. (1975) Biol. Rev. 50,129-165; Cho-Chung, Y. S. (1980) J. Cyclic Nucleotide Res. 6, 163-177;Puck, T. T. (1987) Somatic Cell Mot. Genet. 22, 451-457). Eitherinhibitory or stimulatory effects of cAMP on cell growth have beenreported previously in studies in which cAMP analogs such as N⁶ -O^(2')-dibutyryladenosine 3',5'-cyclic monophosphate or agents that raiseintracellular cAMP to abnormal and continuously high levels were used,and available data are interpreted very differently (Chapowski, F.J.,Kelly, L. A. & Butcher, R. W. (1975) Adv. Cyclic Nucleotide ProteinPhosphorylat. Res. 6, 245-338; Cho-Chung, Y. S. (1979) in Influence ofHormones on Tumor Development, eds. Kellen, J. A. & Hilf, R. (CRC, BocaRaton, Fla.), pp. 55-93); Prasad, K. N. (1981) in The Transformed Cell,eds. Cameron, L. L. & Pool, T. B. (Academic, New York), pp. 235-266;Boynton, A. L. & Whitfield, J. F. (1983) Adv. Cyclic Nucleotide Res. 15,193-294).

Recently, site-selective cAMP analogs were discovered which show apreference for binding to purified preparations of type II rather thantype I cAMP-dependent protein kinase in vitro (Robinson-Steiner, A. M. &Corbin, J. D. (1983) J. Biol. Chem. 258, 1032-1040; greid, D., Ekanger,R., Suva, R. H., Miller, J. P., Sturm, P., Corbin, J. D. & Dskeland, S.O. (1985) Eur. J. Biochem. 150, 219-227), provoke potent growthinhibition, differentiation, and reverse transformation in a broadspectrum of human and rodent cancer cell lines (Katsaros, D., Tortora,G., Tagliaferri, P., Clair, T., Ally, S., Neckers, L., Robins, R. K. &Cho-Chung, Y. S. (1987) FEBS Lett. 223, 97-103; Tortora, G.,Tagliaferri, P., Clair, T., Colamonici, O., Neckers, L. M., Robins, R.K. & Cho-Chung, Y. S. (1988) Blood, 71, 230-233; Tagliaferri,, P.,Katsaros, D., Clair, T., Robins, R. K. & Cho-Chung, Y. S. (1988) J.Biol. Chem. 263, 409-416). The type I and type II protein kinases aredistinguished by their regulatory subunits (RI and RII, respectively)(Corbin, J. D., Keely, S. L. & Park, C. R. (1975) J. Biol. Chem. 250,218-225; Hofmann, F., Beavo, J. A. & Krebs, E. G. (1975) J. Biol. Chem.250, 7795-7801). Four different regulatory subunits [RI.sub.α(previously designated RI) (Lee, D. C., Carmichael, D. F., Krebs, E. G.& McKnight, G. S. (1983) Proc. Natl. Acad. Sci. USA 80, 3608-3612),RI.sub.β (Clegg, C. H., Cadd, G. G. & McKnight, G. S. (1988) Proc. Natl.Acad. Sci. USA 85, 3703-3707), RII.sub.α (RII₅₄) (Scott, J. D., Glaccum,M. B., Zoller, M. J., Uhler, M. D., Hofmann, D. M., McKnight, G. S. &Krebs, E. G. (1987) Proc. Natl. Acad. Sci. USA 84, 5192-5196) andRII.sub.β (RII₅₁) (Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G.,Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schlitz, E.,Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L. & Richards, J.S. (1986) J. Biol. Chem. 261, 12352-12361)] have now been identified atthe gene/mRNA level. Two different catalytic subunits (C.sub.α (Uhler,M. D., Carmichael, D. F., Lee, D.C. Chrivia, J. C., Krebs, E. G. &McKnight, G. S. (1986) Proc. Natl. Acad. Sci. USA 83, 1300-1304) andC.sub.β (Uhler, M. D., Chrivia, J. C. & McKnight, G. S. (1986 ) J. Biol.Chem. 261, 15360-15363; Showers, M. O. & Maurer, R. A. (1986) J. Biol.Chem. 261, 16288-16291)] have also been identified; however,preferential coexpression of either one of these catalytic subunits witheither the type I or type II protein kinase regulatory subunit has notbeen found (Showers, M. O. & Maurer, R. A. (1986) J. Biol. Chem. 261,16288-16291).

The growth inhibition by site-selective cAMP analogs parallels reductionin RI.sub.α with an increase in RII.sub.β, resulting in an increase ofthe RII.sub.β /RI.sub.β ratio in cancer cells (Ally, S., Tortora, G.,Clair, T., Grieco, D., Merlo, G., Katsaros, D., greid, D., Dskeland, S.O., Jahnsen, T. & Cho-Chung, Y. S. (1988) Proc. Natl. Acad. Sci. USA 85,6319-6322; Cho-Chung, Y. S. (1989) J. Natl. Cancer Inst. 81, 982-987).

Such selection modulation of RI.sub.α versus RII.sub.β is not mimickedby treatment with N⁶,O^(2') -dibutyryladenosine 3',5'-cyclicmonophosphate, a previously studied cAMP analog (Ally, S., Tortora, G.,Clair, T., Grieco, D., Merlo, G., Katsaros, D., greid, D., Dskeland, S.O., Jahnsen, T. & Cho-Chung, Y. S. (1988) Proc. Natl. Acad. Sci. USA 85,6319-6322). The growth inhibition further correlates with a rapidtranslocation of RII.sub.β to the nucleus and an increase in thetranscription of the RII.sub.β gene (Ally, S., Tortora, G., Clair, T.,Grieco, D., Merlo, G., Katsaros, D., greid, D., Dskeland, S. O.,Jahnsen, T. & Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. USA 85,6319-6322). These results support the hypothesis that RII.sub.β plays animportant role in the cAMP growth regulatory function (Cho-Chung, Y. S.(1989) J. Natl. Cancer Inst. 81, 982-987).

Antisense RNA sequences have been described as naturally occurringbiological inhibitors of gene expression in both prokaryotes (Mizuno,T., Chou, M-Y, and Inouye, M. (1984), Proc. Natl. Acad. Sci. USA 81,(1966-1970)) and eukaryotes (Heywood, S. M. Nucleic Acids Res. , 14,6771-6772 (1986) and these sequences presumably function by hybridizingto complementary mRNA sequences, resulting in hybridization arrest oftranslation (Paterson, B. M., Roberts, B. E., and Kuff, E. L., (1977)Proc. Natl. Acad. Sci. USA, 74, 4370-4374. Antisenseoligodeoxynucleotides are short synthetic nucleotide sequencesformulated to be complementary to a specific gene or RNA message.Through the binding of these oligomers to a target DNA or mRNA sequence,transcription or translation of the gene can be selectively blocked andthe disease process generated by that gene can be halted. Thecytoplasmic location of mRNA provides a target considered to be readilyaccessible to antisense oligodeoxynucleotides entering the cell; hencemuch of the work in the field has focused on RNA as a target. Currently,the use of antisense oligodeoxynucleotides provides a useful tool forexploring regulation of gene expression in vitro and in tissue culture(Rothenberg, M., Johnson, G., Laughlin, C., Green, I., Craddock, J.,Sarver, N., and Cohen, J. S.(1989) J. Natl. Cancer Inst., 81:1539-1544.

SUMMARY OF THE INVENTION

The invention is related to the discovery that inhibiting the expressionof RI.sub.α in leukemia cells by contact with an antisenseO-oligonucleotides and S-oligonucleotides for RI.sub.α results in theinhibition of proliferation and the stimulation of cell differentiation.Accordingly, the invention is directed to RI.sub.α antisenseoligonucleotides and pharmaceutical compositions thereof for thetreatment of cancer.

In particular, the invention is related to 15- to 30-mer antisenseoligonucleotides which are complementary to a region in the first 100N-terminal codons of RI.sub.α (Seq. ID No:6).

The invention is also related to 15- to 30-mer antisenseoligonucleotides which are a fragment of antisense DNA complementary toRI.sub.α (Seq. ID No: 5).

The invention is also related to pharmaceutical compositions comprisingat least one 15- to 30-mer antisense oligonucleotide which iscomplementary to a region in the first 100 N-terminal codons of RI.sub.α(Seq. ID No:6); and a pharmaceutically acceptable carrier.

The invention is also related to a method for treating cancer bysuppressing growth of cancer cells susceptible to growth suppression andfor inducing cancer cell differentiation in an animal comprisingadministering to an animal in need of such treatment a cancer cellgrowth suppressing amount of an RI.sub.α antisense oligonucleotide.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depicts a graph showing the effect of RI.sub.α antisenseoligodeoxynucleotide (SEQ ID No:1) basal rate of growth of HL-60leukemic cells (A) and the growth of these cells when treated with cAMPanalogs or TPA (B). A, cells were grown (see the Examples) in theabsence (O) or presence (•) of RI.sub.α antisense oligodeoxynucleotide(15 μM). At indicated times, cell counts in duplicate were performed.Data represent the average values ± SD of four experiments. B, On day 4of experiment A, cells exposed or unexposed to RI.sub.α antisenseoligodeoxynucleotide (SEQ ID No:1were reseeded (day 0) at 5×10⁵cells/dish, and cells preexposed to RI.sub.α antisenseoligodeoxynucleotide were further treated with the oligomer at day 0 andday 2. cAMP analogs and TPA were added one time at day 0. Cell countswere performed on a Coulter counter on day 4. 8-Cl, 8-Cl-cAMP (10 μM);8-C1+N.sup. 6 -B, 8-C1-cAMP (5 μM)+N⁶ -benzyl-cAMP (5 μM); TPA (10-⁸ M).The data represent the average values ± SD of four experiments.

"FIG. 2" has been deleted and replaced by "FIGS. 2A, 2B, 2C, 2D, 2E, and2F" depicts a graph showing the effect of RI.sub.α antisenseoligodeoxynucleotide (SEQ ID No:1) on the morphologic transformation ofHL-60 cells. Cells either exposed or unexposed to RI.sub.α antisenseoligodeoxynucleotide were treated with cAMP analogs or TPA as describedin FIG. 1B. On day 4 (see FIG. 1B), cells were washed twice inDulbecco's phosphate-buffered saline and were pelleted onto a glassslide by cytocentrifuge. The resulting cytopreparations were fixed andstained by Wright's stain. ×180.

FIGS. 3A and 3B depicts a Northern blot showing decreased RI.sub.α mRNAexpression in HL-60 leukemic cells exposed to RI.sub.α antisenseoligodeoxynucleotide (SEQ ID No:1) cells were either exposed orunexposed to RI.sub.α antisense oligodeoxynucleotide (15 μM) for 8 hr.Isolation of total RNA and Northern blot analysis followed the methodsdescribed in the Examples. A, ethidium bromide staining of RNA; M,markers of ribosomal RNAS; lanes 1, 2, cells unexposed or exposed toRI.sub.α antisense oligomer. B, Northern blot analysis; the samenitrocellulose filter was hybridized to both RI.sub.α and actin probesin sequential manner. Lanes 1, 2, cells unexposed or exposed to RI.sub.αantisense oligomer.

FIGS. 4A, 4B, and 4C depicts an SDS-PAGE showing the effect of RI.sub.αantisense oligodeoxynucleotide on the basal and induced levels ofRI.sub.α and RII.sub.β cAMP receptor proteins in HL-60 leukemic cells.Cells were either exposed to RI.sub.α antisense oligodeoxynucleotide(SEQ ID No:1) (15 μM) or treated with cAMP analogs as described inFIG. 1. Preparation of cell extracts, the photoactivated incorporationof 8-N₃ -[⁼ P]cAMP and immunoprecipitation using the anti-RI.sub.α oranti-RII.sub.β antiserum and protein A Sepharose, and SDS-PAGE ofsolubilized antigen-antibody complex followed the methods described inthe Examples. Preimmune serum controls were carried out simultaneouslyand detected no immunoprecipitated band. M, ¹⁴ C-labeled marker proteinsof known molecular weight; RI.sub.α, the 48,000 molecular weight RI(Sigma); RII.sub.α, the 56,000 molecular weight RII (Sigma). LanesRII.sub.α and RII.sub.β are from photoaffinity labeling with 8-N₃ -[³²P)cAMP only; lanes 1 to 3, photoaffinity labeling with 8-N₃ -[³² P]cAMPfollowed by immunoprecipitation with anti-RI.sub.α or anti-RII.sub.βantiserum. 8-Cl, 8-Cl-cAMP (5 μM) ; N⁶ -benzyl,N⁶ -benzyl-cAMP (5μM) .The data in the table represent quantification by densitometric scanningof the autoradiograms. The data are expressed relative to the levels incontrol cells unexposed to RI.sub.α antisense oligomer and untreatedwith cAMP analog, which are set equal to 1 arbitrary unit. The datarepresent an average ± SD of three experiments. A and B,immunoprecipitation with anti-RI.sub.α and anti-RII.sub.β antisera,respectively.

FIGS. 5A, 5B, 5C and 5D depicts graphs showing the growth inhibition ofhuman cancer cell lines by RI.sub.α antisense oligodeoxynucleotidehaving SEQ ID No:1 (O-oligo and S-oligo derivatives), compared tocontrols. Cell lines: SK-N-SH, neuroblastoma; LS-174T, colon carcinoma;MCF-7, breast carcinoma; TMK-1, gastric carcinoma. E₂, estradiol-17β.

FIGS. 6A, 6B and 6C depicts the change in morphology of SK-N-SH humanneuroblastoma cells exposed to RI.sub.α antisense oligodeoxynucleotidehaving SEQ ID No:1.

FIGS. 7A and 7B depicts a graph showing that RI.sub.α antisenseoligodeoxynucleotide and its phosphorothioate analog (SEQ ID No:1)inhibit the in vivo growth of LS-174T human colon carcinoma in athymicmice. FIG. 7A shows the oligodeoxynucleotide concentration-dependentinhibition of tumor growth. O-oligo, RI.sub.α antisenseoligodeoxynucleotide; S-oligo, phosphorothioate analog of RI.sub.αantisense oligomer. The cholesterol pellets (total weight 20 mg)containing the indicated doses of O-oligo or S-oligo were implanted s.c. one time, at zero time, and tumor sizes were measured. Tumor volume(see Materials and Methods, Example 3) represents an average ± S.D. of 7tumors. FIG. 7B shows the temporal effect of antisenseoligodeoxynucleotide phosphorothioate analogs on tumor growth. S-oligosas indicated at 0.3 mg dose in cholesterol pellets (total weight 20 mg)were implanted s.c. 2×/week, and tumor volume (see Materials andMethods, Example 3) represents an average ± S.D. of 7 tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Antisense therapy is the administration of exogenous oligonucleotideswhich bind to a target polynucleotide located within the cells. The term"antisense" refers to the fact that such oligonucleotides arecomplementary to their intracellular targets, e.g., RI.sub.α. See forexample, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of GeneExpression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The RI.sub.αantisense oligonucleotides of the present invention include derivativessuch as S-oligonucleotides (phosphorothioate derivatives or S-oligos,see, Jack Cohen, supra) which exhibit enhanced cancer cell growthinhibitory action (see FIGS. 5 and 7A).

S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfurtransfer reagent. See Iyer, R.P. et al., J. Org. Chem. 55:4693-4698(1990) ; and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990),the disclosures of which are fully incorporated by reference herein.

The RI.sub.α antisense oligonucleotides of the present invention may beRNA or DNA which is complementary to and stably hybridizes with thefirst 100 N-terminal codons of the RI.sub.α genome or the correspondingmRNA. Use of an oligonucleotide complementary to this region allows forthe selective hybridization to RI.sub.α mRNA and not to mRNA specifyingother regulatory subunits of protein kinase. Preferably, the RI.sub.αantisense oligonucleotides of the present invention are a 15 to 30-merfragment of the antisense DNA molecule having SEQ ID NO:5 whichhybridizes to RI.sub.α mRNA. Alternative-ly, RI.sub.α antisenseoligonucleotide is a 15- to 30-mer oligonucleotide which iscomplementary to a region in the first 100 N-terminal codons of RI.sub.α(Seq. ID No:6). Most preferably, the RI.sub.α antisense oligonucleotidehas SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, or SEQ ID No:4.

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one of theRI.sub.α antisense oligonucleotides of the invention in combination witha pharmaceutically acceptable carrier. In one embodiment, a singleRI.sub.α antisense oligonucleotide is utilized. In another embodiment,two RI.sub.α antisense oligonucleotides are utilized which arecomplementary to adjacent regions of the RI.sub.α genome. Administrationof two RI.sub.α antisense oligonucleotides which are complementary toadjacent regions of the RI.sub.α genome or corresponding mRNA may allowfor more efficient inhibition of RI.sub.α genomic transcription or mRNAtranslation, resulting in more effective inhibition of cancer cellgrowth.

Preferably, the RI.sub.α antisense oligonucleotide is coadministeredwith an agent which enhances the uptake of the antisense molecule by thecells. For example, the RI.sub.α antisense oligonucleotide may becombined with a lipophilic cationic compound which may be in the form ofliposomes. The use of liposomes to introduce nucleotides into cells istaught, for example, in U.S. Pat. Nos. 4,897,355 and 4,394,448, thedisclosures of which are incorporated by reference in their entirety.See also U.S. Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788,4,673,567, 4,247,411, 4,814,270 for general methods of preparingliposomes comprising biological materials.

Alternatively, the RI.sub.α antisense oligonucleotide may be combinedwith a lipophilic carrier such as any one of a number of sterolsincluding cholesterol, cholate and deoxycholic acid. A preferred sterolis cholesterol.

In addition, the RI.sub.α antisense oligonucleotide may be conjugated toa peptide that is ingested by cells. Examples of useful peptides includepeptide hormones, antigens or antibodies, and peptide toxins. Bychoosing a peptide that is selectively taken up by the neoplastic cells,specific delivery of the antisense agent may be effected. The RI,antisense oligonucleotide may be covalently bound via the 5'H group byformation of an activated aminoalkyl derivative. The peptide of choicemay then be covalently attached to the activated RI.sub.α antisenseoligonucleotide via an amino and sulfhydryl reactive hetero bifunctionalreagent. The latter is bound to a cysteine residue present in thepeptide. Upon exposure of cells to the RI.sub.α antisenseoligonucleotide bound to the peptide, the peptidyl antisense agent isendocytosed and the RI.sub.α antisense oligonucleotide binds to thetarget RI.sub.α mRNA to inhibit translation. See PCT ApplicationPublication No. PCT/US89/02363.l

"The RI-alpha antisense oligonucleotides of the invention inhibit thegrowth of HL-60 cells, human colon carcinoma LS-174T, neuroblastomacells, breast carcinoma, and gastric carcinoma cells in vitro. Inaddition, these antisense oligomers can inhibit the growth of humancarcinoma LS-174T cells transplanted into athymic mice."

The RI.sub.α antisense oligonucleotides and the pharmaceuticalcompositions of the present invention may be administered by any meansthat achieve their intended purpose. For example, administration may beby parenteral, subcutaneous, intravenous, intramuscular,intra-peritoneal, or transdermal routes. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

Compositions within the scope of this invention include all compositionswherein the RI.sub.α antisense oligonucleotide is contained in an amountwhich is effective to achieve inhibition of proliferation and/orstimulate differentiation of the subject cancer cells. While individualneeds vary, determination of optimal ranges of effective amounts of eachcomponent is with the skill of the art. Typically, the RI.sub.αantisense oligonucleotide may be administered to mammals, e.g. humans,at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated.

In addition to administering the RI.sub.α antisense oligonucleotides asa raw chemical in solution, the RI.sub.α antisense oligonucleotides maybe administered as part of a pharmaceutical preparation containingsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the RI.sub.α antisenseoligonucleotide into preparations which can be used pharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the RI.sub.α antisense oligonucleotides in water-solubleform, for example, water-soluble salts. In addition, suspensions of theactive compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides. Aqueous injection suspensionsmay contain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

The antisense oligonucleotides of the present invention may be preparedaccording to any of the methods that are well known to those of ordinaryskill in the art. Preferably, the antisense oligonucleotides areprepared by solid phase synthesis. See, Goodchild, J., BioconjugateChemistry, 1:165-167 (1990), for a review of the chemical synthesis ofoligonucleotides. Alternatively, the antisense oligonucleotides can beobtained from a number of companies which specialize in the customsynthesis of oligonucleotides.

Having now generally described this invention, the same will beunderstood by reference to an example which is provided herein forpurposes of illustration only and is not intending to be limited unlessotherwise specified. The entire text of all applications, patents andpublications, if any, cited above and below are hereby incorporated byreference.

EXAMPLES EXAMPLE 1

Oligodeoxynuclootides. The 21-mer oligodeoxynucleotides used in thepresent studies were synthesized at Midland Certified Reagent Co.(Midland, Tex.) and had the following sequences: human RI.sub.α(Sandberg, M., Tasken, K., Oyen, O., Hansson, V. & Jahnsen, T. (1987)Biochem. Biophys. Res. Commun., 149, 939-945) antisense,5'-GGC-GGT-ACT-GCC-AGA-CTC-CAT-3' (SEQ ID No:1); human RII.sub.β (Levy,F. O., Oyen, O., Sandberg, M., Tasken, K., Eskild, W., Hansson, V. &Jahnsen, T. (1988) Mol. Endocrinol., 2, 1364-1373) antisense5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3'; human RII.sub.β (Oyen, O., Myklebust,F., Scott, J. D., Hansson, V. & Jahnsen, T. (1989) FEBS Lett. 246,57-64) antisense, 5'-CGG-GAT-CTG-GAT-GTG-GCT-CAT-3'; and the randomsequence oligodeoxynucleotide was made of a mixture of all fournucleotides at every position.

Cell Growth Experiment. Cells grown in suspension culture in RPM1 1640medium supplemented with 10% heat-inactivated fetal bovine serum,penicillin (50 U/ml), streptomycin (500 μg/ml), and 1 mM glutamine(Gibco, Grand Island, N.Y.) were seeded at 5×10⁵ cells per dish.Oligodeoxynucleotides were added after seeding and every 48 hrthereafter. Cell counts were performed on a Coulter counter. Cellsunexposed or exposed to oligodeoxynucleotides for 4 days were reseeded(day 0) at 5×10⁵ cells/dish, and cells preexposed to theoligodeoxynucleotide were further treated with the oligomer at day 0 andday 2. cAMP analogs (kindly provided by Dr. R. K. Robins, Nucleic AcidResearch Institute, Costa Mesa, Calif.) or12-O-tetradecanoylphorbol-13-acetate (TPA) were added one time at day 0.Cell counts were performed on day 4.

Immunoprecipitation of RIα and RII.sub.β cAMP Receptor Proteins afterPhotoaffinity Labeling with 8-N₃ -[³² P]cAMP. Cell extracts wereprepared at 0°-40° C. The cell pellets (2×10⁶ cells), alter two washeswith PBS, were suspended in 0.5 ml buffer Ten (0.1M NaCl, 5 mM MgCl₂, 1%Nonidet P-40, 0.5% Na deoxycholate, 2 KIU/ml bovine aprotinin, and 20 mMTris-HC1, pH 7.4) containing proteolysis inhibitors (Tortora, G., Clair,T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci. USA 87, 705-708),vortex-mixed, passed through a 22-gauge needle 10 times, allowed tostand for 30 min at 4° C., and centrifuged at 750×g for 20 min; theresulting supernatants were used as cell lysates. The photoactivatedincorporation of 8-N₃ -[³² P]cAMp (60.0 Ci/mmol), and theimmunoprecipitation using the anti-RI.sub.α or anti-RII.sub.β antiserum(kindly provided by Dr. S. O. Dskeland, University of Bergen, Bergen,Norway) and protein A Sepharose and SDS-PAGE of solubilizedantigen-antibody complex followed the method previously described(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen,T. & Dskeland, S. O. (1985) J. Biol. Chem. 260, 3393-3401)..

cAMP-Dependent Protein Kinase Assays. After two washes with Dulbecco'sphosphate-buffered saline, cell pellets (2×10⁶ cells) were lysed in 0.5ml of 20 mM Tris (pH 7.5), 0.1 mM sodium EDTA, 1 mM dithiothreitol, 0.1mM pepstatin, 0.1 mM antipain, 0.1 mM chymostatin, 0.2 mM leupeptin, 0.4mg/ml aprotinin, and 0.5 mg/ml soybean trypsin inhibitor, using 100strokes of a Dounce homogenizer. After centrifugation (Eppendorf 5412)for 5 min, the supernatants were adjusted to 0.7 mg protein/ml andassayed (Uhler, M. D. & McKnight, G. S. (1987) J. Biol. Chem. 262,15202-15207) immediately. Assays (40 μl total volume) were performed for10 min at 300° C. and contained 200 μM ATP, 2.7×10⁶ cpm γ[³² P]ATP, 20mM MgCl₂, 100 μM Kemptide (Sigma K-1127) (Kemp, B. E., Graves, D. J.,Benjamin, E. & Krebs, E. G. (1977) J. Biol. Chem. 252, 4888-4894), 40 mMTris (pH 7.5), ± 100 μM protein kinase inhibitor (Sigma P-3294) (Cheng,H.-C., Van Patten, S. M., Smith, A. J. & Walsh, D. A. (1985) Biochem. J.231, 655-661), ± 8 μM cAMP and 7 μg of cell extract. The phosphorylationof Kemptide was determined by spotting 20 μl of incubation mixture onphosphocellulose filters (Whatman, P81) and washing in phosphoric acidas described (Roskoski, R. (1983) Methods Enzymol. 99, 3-6) .Radioactivity was measured by liquid scintillation using Econofluor-2(NEN Research Products NEF-969).

Isolation of Total RNA and Northern Blot Analysis. The cells (10⁸ washedtwice with phosphate-buffered saline) were lysed in 4.2M guanidineisothiocyanate containing 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl(N-lauroylsarcosine Na⁺), and 0.1M β-mercaptoethanol, and the lysateswere homogenized, and total cellular RNA was sedimented through a CsClcushion (5.7M CsCl, 10 mM EDTA) as described by Chirgwin et at.(Chirgwin, J. M., Przybyla, A. E., MacDonald, R. Y. & Rutter, W. J.(1977) Biochemistry 18, 5284-5288). Total cellular RNA containing 20 mM3-[N-morpholine)propane-sulfonic acid (pH 7.0), 50% formamide, and 6%formaldehyde was denatured at 65° C. for 10 min and electrophoresedthrough a denaturing 1.2% agarose-2.2M formaldehyde gel. The gels werethen transferred to Biotrans nylon membranes (ICN Biomedicals) by themethod of Thomas (Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77,5201-5205) and hybridized to the following two ³² P-labelednick-translated CDNA probes: 1.5 kilobase (kb) CDNA clone containing theentire coding region for the human cAMP-dependent protein kinase type Iregulatory subunit, RI.sub.α (Sandberg, M., Tasken, K., Oyen, O.,Hansson, V. & Jahnsen, T. (1987) Biochem. Biophys. Res. Commun. 149,939-945) (kindly provided by Dr. T. Jahnsen, Institute of Pathology,Rikshospitalet, Oslo, Norway), and human β actin (oncor p7000 β actin).

RESULTS

The RI.sub.α antisense oligodeoxynucleotide at 15 μM concentration hadimmediate effects on the rate of proliferation of HL-60 cells. By 4-5days in culture, while cells unexposed to RI.sub.α antisense oligomerdemonstrated an exponential rate of growth, cells exposed to theantisense oligomer exhibited a reduced growth rate and eventuallystopped replicating (FIG. 1A). This inhibitory effect on cellproliferation persisted throughout the culture period. The growthinhibition was not due to cell killing; cells were over 90% viable afterexposure to RI.sub.α antisense oligomer (15 μM) f or 7 days as assessedby f low cytometry using forward and side scatter. RI.sub.α sense,RII.sub.α or RII.sub.β antisense, or a random sequenceoligodeoxynucleotide had no such growth inhibitory effect.

Cells unexposed or exposed to RI.sub.α antisense oligodeoxynucleotidefor 4 days in culture were reseeded and examined for their response totreatment with cAMP analogs or TPA. In cells unexposed to RI.sub.αantisense oligodeoxynucleotide, 8-Cl-cAMP (10 μM) produced 60% growthinhibition, and 80% growth inhibition was achieved by 8-Cl-cAMP (5 μM)plus N⁶ -benzyl-cAMP (5 μM) (FIG. 1B) (Tortora, G., Tagliaferri, P.,Clair, T., Colamonici, O. Neckers, L. M., Robins, R. K. & Cho-Chung, Y.S. (1988) Blood 71, 230-233), and TPA (10⁻⁸ M) exhibited 60% growthinhibition (FIG. 1B). In contrast, cells exposed to antisenseoligodeoxynucleotide exhibited retarded growth (25% the rate of growthof cells unexposed to the antisense oligomer) and neither cAMP analogsnor TPA brought about further retardation of growth (FIG. 1B).

HL-60 cells undergo a monocytic differentiation upon treatment withsite-selective cAMP analogs. Cells either unexposed or exposed toRI.sub.α antisense oligodeoxynucleotide were examined for theirmorphology before and alter treatment with cAMP analogs. As shown inFIG. 2, in cells unexposed to RI.sub.α antisense oligomer, 8-Cl-cAMPplus N⁶ -benzyl-cAMP induced a monocytic morphologic changecharacterized by a decrease in nuclear-to-cytoplasm ratio, abundantruffled and vacuolated cytoplasm, and loss of nucleoli. Strikingly, thesame morphologic change was induced when cells were exposed to RI.sub.αantisense oligodeoxynucleotide (FIG. 2). Moreover, the morphologicchanges induced by antisense oligomer were indistinguishable from thatinduced by TPA (FIG. 2).

To provide more evidence that the growth inhibition and monocyticdifferentiation induced in HL-60 cells exposed to the RI.sub.α antisenseoligodeoxynucleotide were due to an intracellular effect of theoligomer, the RI.sub.α mRNA level was determined. As shown in FIG. 3,3.0 kb RI.sub.α mRNA (Sandberg, M., Tasken, K., Oyen, O., Hansson, V. &Jahnsen, T. (1987) Biochem. Bioshys. Res. Commun. 149, 939-945) wasvirtually undetectable in cells exposed for 8 hr to RI.sub.α antisenseoligodeoxynucleotide (FIG. 3B, lane 2), and the decrease in RI.sub.αmRNA was not due to a lower amount of total RNA as shown by the ethidiumbromide staining (compare lane 2 with lane 1 of FIG. 3A). Conversely, anenhanced level of actin mRNA was detected in cells exposed to RI.sub.αantisense oligomer (FIG. 3B). Whether the increase in actin mRNA levelrepresents changes in cytoskeletal structure is not known.

The levels of cAMP receptor proteins in these cells was then determinedby immunoprecipitation using anti-RI.sub.α and anti-RII.sub.β antisera(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen,T. & Dskeland, S. O. (1985) J. Biol. Chem. 260, 3393-3401) afterphotoaffinity labeling of these receptor proteins with 8-N₃ -[³² P]cAmp.In control cells, treatment with 8-Cl-cAMP plus N⁶ -benzyl-cAMP broughtabout a 70% reduction in RI.sub.α with a 3-fold increase in RII.sub.β,resulting in a 10-fold increase in the ratio of RII.sub.β, (FIG. 4)(Cho-Chung, Y. S. (1989) J. Natl. Cancer Inst. 81, 982-987). Exposure ofthese cells to RI.sub.α antisense oligodeoxynucleotide for 4 daysbrought about marked changes in both and RI.sub.α and RII.sub.β levels;an 80% reduction in RI.sub.α with a 5-fold increase in RII.sub.βresulted in a 25-fold increase in the ratio of RII.sub.β /RI.sub.αcompared with that in control cells (FIG. 4). Since growth inhibitionand differentiation were appreciable after 3-4 days of exposure toRI.sub.α antisense oligomer, the changing levels of RI.sub.α andRII.sub.β proteins appears to be an early event necessary for commitmentto differentiation.

Data in FIG. 4 showed that suppression of RI.sub.α by the antisenseoligodeoxynucleotide brought about a compensatory increase in RII.sub.βlevel. Such coordinated expression of RI and RII without changes in theamount of C subunit has been shown previously (Hofman, F., Bechtel, P.J. & Krebs, E. G. (1977) J. Biol. Chem. 252, 1441-1447; Otten, A. D. &Mcknight, G. S. (1989) J. Biol. Chem. 264, 20255-20260). The increase inRII.sub.β may be responsible for the differentiation induced in thesecells after exposure to RI.sub.β antisense oligodeoxynucleotide. Theincrease in RII.sub.β mRNA or RII.sub.β protein level has beencorrelated with cAMP analog-induced differentiation in K-562 chronicmyelocytic leukemic cells (Tortora, G., Clair, T., Katsaros, D., Ally,S., colamonici, O., Neckers, L. M., Tagliaferri, P., Jahnsen, T.,Robins, R. K. & Cho-Chung, Y. S. (1989) Proc. Natl. Acad. Sci. USA 86,2849-2852) and in erythroid differentiation of Friend erythrocyticleukemic cells (Schwartz, D. A. & Rubin, C. S. (1985) J. Biol. Chem.260, 6296-6303). In a recent report (Tortora, G., Clair, T. & Cho-Chung,Y. S. (1990) Proc. Natl. Acad. Sci. USA 87, 705-708), we have provideddirect evidence that RII.sub.β is essential for the cAMP-induceddifferentiation in HL-60 cells. HL-60 cells that were exposed toRII.sub.β antisense oligodeoxynucleotide became refractory to treatmentwith cAMP analogs and continued to grow.

The essential role of RII.sub.β in differentiation of HL-60 cells wasfurther demonstrated when these cells were exposed to both RI.sub.α andRII.sub.β antisense oligodeoxynucleotides simultaneously. As shown inTable 1, RI.sub.α antisense oligodeoxynucleotide (SEQ ID No:1) induced amarked increase in the expression of monocytic surface antigens [Leu 15(Landay, A., Gartland, L. & Clement, L. T. (1983) J. Immunol. 131,2757-2761) and Leu M3 (Dimitriu-Bona, A., Bummester, G. R., Waters, S.J. & Winchester, R. J. (1983) J. Immunol. 130, 145-152)] along with adecrease in markers related to the immature myelogenous cells (My9(Talle, M. A., Rao, P. E., Westberg, E., Allegar, N., Makowski, M.,Mittler, R. S. & Goldstein, G. (1983) Cell. Immunol. 78, 83.; Todd, R.F. III, Griffin, J. D., Ritz, J., Nadler, L. M. Abrams, T. & Schlossman,S. F. (1981) Leuk. Res. 5, 491)). These changes in surface markerexpression were abolished when cells were exposed simultaneously to bothand RI.sub.α and RII.sub.β antisense oligodeoxynucleotides (Table 1) .RII.sub.α cAMP receptor was not detected in HL-60 cells (Cho-Chung, Y.S., Clair, T., Tagliaferri, P., Ally, S., Katsaros, D., Tortora, G.,Neckers, L., Avery, T. L., Crabtree, G. W. & Robins, R. K. (1989) CancerInvest. 7(2), 161-177), and RII.sub.α antisense oligodeoxynucleotideshowed no interference with the effects of RI.sub.α antisense oligomer(Table 1).

Cells exposed to both RI.sub.α and RII.sub.β antisenseoligodeoxynucleotides were neither growth inhibited nor differentiatedregardless of cAMP analog treatment. We interpret these results toreflect the blockage of cAMP-dependent growth regulatory pathway. Cellsunder these conditions are no longer cAMP-dependent but survive andproliferate probably through an alternate pathway. Thus, suppression ofboth RI.sub.α and RII.sub.β gene expression led to an abnormal cellulargrowth regulation similar to that in mutant cell lines (Gottesman, M. M.(1980) Cell 22, 329-330), those that contain either deficient ordefective regulatory subunits of cAMP-dependent protein kinase and areno longer sensitive to cAMP stimulus.

Our results demonstrated that cAMP transduces signals for dual controls,either positive or negative, on cell proliferation, depending on theavailability of RI.sub.α or RII.sub.β receptor proteins. The RI.sub.αantisense oligodeoxynucleotide which brought about suppression ofRI.sub.α along with enhancement of RII.sub.β expression led to terminaldifferentiation of HL-60 leukemia with no sign of cytotoxicity.

It is unlikely that free C subunit increase in cells exposed to RI.sub.αantisense oligodeoxynucleotide was responsible for the differentiation,because cells exposed to RII.sub.β antisense or both RI.sub.α andRII.sub.β antisense oligodeoxynucleotides, conditions which also wouldproduce free C subunit, continued to grow and became refractory to cAMPstimulus. In order to directly verify this we measuredphosphotransferase activity in cells that are exposed or unexposed tothe antisense oligodeoxynucleotides using kemptide (Kemp, B. E., Graves,D. J., Benjamin, E. & Krebs, E. G. (1977) J. Biol. Chem. 252, 4888-4894)as a substrate in the presence and absence of a saturating concentrationof cAMP and in the presence and absence of the heat-stable proteinkinase inhibitor (Cheng, H.-C., Van Patten, S. M., Smith, A. J. & Walsh,D. A. (1985) Biochem. J. 231, 655-661). This method of assay givesaccurate determination of the relative levels of dissociated C and totalC activity. Cell extracts from untreated HL-60 cells exhibited a verylow level of dissociated C and were stimulated 36-fold by cAMP (Table2). This cAMP-stimulated activity was almost completely inhibited by theheat-stable protein kinase inhibitor (Table 2), indicating that thetotal C activity measured was cAMP-dependent protein kinase. In cellsexposed to RI.sub.α antisense, RII.sub.β antisense, or RI.sub.α andRII.sub.β antisense oligodeoxynucleotide, the free C activity was notincreased as compared to unexposed control cells, although there was asmall difference in the total cAMP-stimulated activity (Table 2). Theseresults provide direct evidence that free catalytic subunit is notresponsible for the differentiation observed in HL-60 cells.

Over expression of RI.sub.α cAMP receptor protein has also been found inthe majority of human breast and colon primary carcinomas examined(Bradbury, A. W., Miller, W. R., Clair, T., Yokozaki, H. & Cho-Chung, Y.S. (1990) Proc. Am. Assoc. Cancer Res. 31, 172), suggesting an importantin vivo role of cAMP receptor in tumor growth as well. However, theprecise role of RI.sub.α in cell proliferation is not known at present.RI.sub.α may suppress RII.sub.β production by titrating out C subunit,or it may be a transducer of mitogenic signals leading to cellproliferation. Our results demonstrate that RI.sub.α antisenseoligodeoxynucleotide provides a useful genetic tool for studies on therole of cAMP receptor proteins in cell proliferation anddifferentiation, and contribute to a new approach in the control ofmalignancy.

    ______________________________________                                                       Surface Makers                                                 Treatment        Leu15      LeuM3   My9                                       ______________________________________                                        Control          10         2       100                                       RI.sub.α  antisense                                                                      80         98       80                                       RI.sub.α  antisense + RII.sub.β  antisense                                          11         2       100                                       RII.sub.β  antisense                                                                      13         3       100                                       RI.sub.α  antisense + RII.sub.α  antisense                                         85         100      80                                       ______________________________________                                    

Surface antigen analysis was performed by flow cytometry usingmonoclonal antibodies reactive with either monocytic or myeloid cells.The monoclonal antibodies used were Leu 15, Leu M3, and My9. 2×10⁴ cellswere analyzed for each sample, and cell gating was performed usingforward and side scatter. The numbers represent % positive and representthe average values of three experiments.

                                      TABLE 2                                     __________________________________________________________________________    Protein kinase activity in HL-60 cells                                                Activity                                                                            Relative                                                                            Activity                                                                             Relative                                                                            Stimulation                                  Treatment                                                                             -cAMP to control                                                                          +cAMP  to control                                                                          (fold)                                       __________________________________________________________________________    -PKI                                                                          Control 23.0 ± 6.6                                                                       1.0   837 ± 87                                                                          1.0   36                                           RI.sub.α  antisense                                                             22.9 ± 5.4                                                                       1.0   944 ± 18                                                                          1.1   41                                           RII.sub.β  antisense                                                             22.8 ± 8.1                                                                       1.0   1,028 ± 154                                                                       1.2   45                                           RI.sub.α  and                                                                   24.3 ± 7.0                                                                       1.1   802 ± 36                                                                          1.0   33                                           RII.sub.β  antisense                                                     +PKI                                                                          Control 17.5 ± 8.7                                                                       1.0   37.0 ± 8.4                                                                        1.0   2.1                                          RI.sub.α  antisense                                                             25.0 ± 8.8                                                                       1.4   22.6 ± 8.8                                                                        0.6   0.9                                          RII.sub.β  antisense                                                             24.0 ± 2.6                                                                       1.4   24.8 ± 3.9                                                                        0.7   1.0                                          RI.sub.α  and                                                                   19.0 ± 5.9                                                                       1.1   19.1 ± 8.2                                                                        0.5   1.0                                          RII.sub.β  antisense                                                     __________________________________________________________________________

Cells were exposed to each of 15 μM concentrations of RI.sub.α,RII.sub.β, or RI.sub.α and RII.sub.β antisense oligodeoxynucleotide for4 days as shown in FIG. 1A. The data represent an average ± SD ofduplicate determinations of three identical experiments. *Picomolesphosphate transferred to Kemptide per min/mg protein.

EXAMPLE 2

Next, the RI.sub.α antisense oligonucleotide having SEQ ID NO:1 wasadministered to mice having an experimental tumor. A pellet of RI.sub.αantisense oligonucleotide (25 mg/Kg) and cholesterol (1000 mg/Kg) wasimplanted s.c. in the left flank of athymic mice which had been injectedin the right flank with LS-174T human colon cancer cells (2×10⁶ cells)suspended in phosphate-buffered saline. Tumor measurements and mouseweights were recorded on the initial day of treatment (staging day), andat the end of treatment (staging day +5). The mean tumor weight change(Δ), was based on length and width measurements in millimeters. After afew days, the tumor growth was inhibited when compared to control cells(see Table 3). No change in body weight was noted in the control andtreated animals.

                  TABLE 3                                                         ______________________________________                                        Effect of RI.sub.α  antisense oligodeoxynucleotide                      s.c. pellet on the growth of LS-174T human                                    color carcinoma in athymic mice                                               Treatment.sup.a                                                                             Initial mean.sup.c                                                                       Final mean.sup.d                                     s.c. pellet   tumor wt   tumor wt   %                                         implanted     (mg)       (mg)       ΔT/ΔC.sup.e                   ______________________________________                                        Control       25         450        --                                        RI.sub.α  antisense                                                                   25         230        48                                        (0.5 mg)                                                                      8-Cl cAMP (1 mg).sup.b +                                                                    34         250        51                                        N.sup.6 benzyl cAMP (1 mg)                                                    ______________________________________                                         .sup.a 20 mg pellet lyophilized consisting of indicated doses of              RI.sub.α  antisense or cAMP analogs plus supplement doses of            cholesterol.                                                                  .sup.b The growth inhibitory effect of these cAMP analogs correlate with      decrease in RI.sub.α  (Natl. Cancer Inst. 81 982 (1989)) and is         shown here for comparison.                                                    .sup.c Mean tumor weight per group (4 mice) on staging day.                   .sup.d Mean tumor weight per group on staging day +5.                         .sup.e % of change in test tumor weight (ΔT)/change in control tumo     weight (ΔC).                                                       

In other in vitro experiments, the RI.sub.α antisense oligonucleotidehaving SEQ ID NO:1 was added to dishes containing neuroblastoma, coloncarcinoma, breast carcinoma and gastric carcinoma cells. As shown inFIG. 5, the RI.sub.α antisense oligonucleotide having SEQ ID No: 1inhibited proliferation of all cancer cell types when compared tocontrol cells. Moreover, the RI.sub.α antisense oligonucleotide havingSEQ ID No: 1 caused differentiation of the human neuroblastoma cells(see FIG. 6).

EXAMPLE 3

Next, the effect of 0-oligo and S-oligo RI.sub.α antisenseoligonucleotides on the growth of LS-174T human colon carcinoma inathymic mice was compared.

Materials and Methods. We synthesized (Milligen Biosearch 8700 DNAsynthesizer (Bedford, MA)]the 21-mer antisense oligodeoxynucleotides andtheir phosphorothioate analogs complementary to the human RI.sub.α,human RII.sub.β mRNA transcripts starting from the first codon, andmismatched sequence (random) oligomers of identical size. The oligomershad the following sequences: RI.sub.α antisense,5'-GGC-GGT-ACT-GCC-AGA-CTC-CAT-3' (SEQ ID No:1); RII.sub.β antisense,5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3'; and random oligo,5'-CGA-TCG-ATC-GAT-CGA-TCG-TAC-3'.

LS-174T human colon carcinoma cells (2×10⁶) were injected s.c. inathymic mice, and the antisense oligodeoxynucleotides in the form ofeither a cholesterol pellet or 50% sesame oil emulsion were administereds.c. 1 week later when mean tumor sizes usually were 25-50 mg. Tumorvolume was based on length and width measurements and calculated by theformula 4/3 πr³, where r=(length+width)/4.

Results and Discussion. FIG. 7 shows the dose-and and time-dependenteffect of an RI.sub.α antisense oligodeoxynucleotide (O-oligo) at 0.2and 0.5 mg doses in cholesterol pellets administered s.c. one time (atzero time); it brought about 20 and 46% growth inhibition, respectively,in 7 days when compared with control (untreated) tumors (FIG. 7A).Strikingly, the RI.sub.α antisense phosphorothioate analog (S-oligo) ata 0.2 mg dose (cholesterol pellet, s.c.) gave a 60% growth inhibition atday 7, exhibiting a 3-fold greater potency than the O-oligo antisense(FIG. 7A). The growth inhibitory effect of RI.sub.α antisense S-oligowas even greater when animals were treated for a longer period. TheRI.sub.α antisense S-oligo at a 0.3 mg dose in a cholesterol pellet, 2times/week s.c. implantation for 3 weeks, resulted in a 80% growthinhibition; the tumor growth almost stopped after 2 weeks of treatment(FIG. 7B). RI.sub.α antisense O-oligo or S-oligo administered s.c. as50% sesame oil emulsion gave similar results. RI.sub.α antisense S-oligobrought about no apparent toxicity in animals; no body weight loss orother toxic symptoms were observed during the 3 weeks of treatment.

The growth inhibitory effect brought about by RI.sub.α antisense S-oligowas the specific effect of the oligomer: RII.sub.β antisense or random(mismatched sequence) S-oligos of the identical size as the RI.sub.αantisense oligomer has no effect on the tumor growth (FIG. 7B).

To provide more evidence that the growth inhibition observed in coloncarcinomas in athymic mice treated with RI.sub.α antisenseoligodeoxynucleotide was due to an intracellular effect of the oligomer,the levels of RI.sub.α and RII.sub.β cAMP receptor proteins in thesetumors were determined. RI.sub.α levels were determined byimmunoblotting (Ally, S., Proc., Natl. Acad. Sci. USA 85:6319-6322(1988)) using monoclonal antibody against human RI.sub.α (kindlyprovided by Drs. T. Lea, University of Oslo, Oslo, Norway, and S. O.Dskeland, University of Bergen, Bergen, Norway), and RII.sub.β wasmeasured by immunoprecipitation (Tortora, G., et al., Proc. Natl. Acad.Sci. USA 87:705-708 (1990)) with anti-RII.sub.β antiserum (kindlyprovided by Dr. S. O. Dskeland) after photoaffinity labeling ofRII.sub.β with [³² P] 8-N₃ -cAMP. As shown in Table 4, RI.sub.αantisense S-oligomer treatment brought about a marked reduction (80%decrease) of RI.sub.α level in tumors as compared with that in untreatedcontrol tumors. This suppression of RI.sub.α expression by RI.sub.αantisense S-oligomer brought about a 2-fold increase in RII.sub.β level(Table 4). Such coordinated expression of RI.sub.α and RII.sub.β withoutchanges in the amount of catalytic subunit of protein kinase has beenshown in HL-60 leukemia cells that demonstrated growth inhibition anddifferentiation upon exposure to RI.sub.α antisenseoligodeoxynucleotide. On the other hand, a 50% increase in RI.sub.αlevel along with 80% suppression in RII.sub.β level was observed intumors after treatment with RII.sub.β antisense S-oligomer (Table 4)which had no effect on tumor growth (FIG. 7). Random (mismatchedsequence) S-oligomer which had no effect on tumor growth (FIG. 7) alsoshowed no effect on RI.sub.α levels (Table 4). Thus, reduction inRI.sub.α expression appears to trigger a decrease or halt in tumorgrowth upon treatment with RI.sub.α antisense oligomer. Our resultsdemonstrated that cAMP transduces signals for dual control, eitherpositive or negative, on cell proliferation, depending on theavailability of RI.sub.α or RII.sub.β receptor proteins. The RI.sub.αantisense oligodeoxynucleotide, which suppressed RI.sub.α and enhancedRII.sub.β expression, led to inhibition of in vivo growth of solid coloncarcinoma in athymic mice with no symptoms of toxicity in animals. Thephosphorothioate analog (S-oligo) of RI.sub.α antisense oligomerexhibited a greater potency than the antisense of unmodifiedoligodeoxynucleotide (O-oligo). It has been shown that S-oligos, ascompared with O-oligos, more readily enter cells, are more resistant toendonucleases, and yet exhibit high efficacy in hybridization withtarget mRNAs or DNAs (Stein, C. A., et al., In: J. S. Cohen (ed.),Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, pp.97-117. Boca Raton, Fla. CRC Press, Inc. (1989)).

These results demonstrate here for the first time the striking in vivoeffect of antisense oligodeoxynucleotide in the suppression ofmalignancy. The depletion of RI.sub.α, the type I regulatory subunit ofcAMP-dependent protein kinase, by means of an antisenseoligodeoxynucleotide, especially with its phosphorothioate analog, leadsto a successful halt of tumor growth in vivo with no symptoms oftoxicity, suggesting great potential of this antisenseoligodeoxynucleotide for clinical application.

                  TABLE 4                                                         ______________________________________                                        Suppression of RI.sub.α  cAMP Receptor Expression by                    RI.sub.α                                                                Antisense Oligodeoxynucleotide (S-oligo); SEQ ID NO: 1 Results                in Compensatory Increase in RII.sub.α  Receptor                                      Relative Levels                                                  Treatment      RI.sub.α                                                                          RII.sub.β                                       ______________________________________                                        None           1.0 ± 0.1                                                                            1.0 ± 0.1                                         RI.sub.α  0.2 ± 0.03                                                                          2.0 ± 0.2                                         S-oligo                                                                       RII.sub.β 1.5 ± 0.2                                                                             0.2 ± 0.02                                       S-oligo                                                                       Random S-oligo 1.0 ± 0.1                                                                            1.0 ± 0.1                                         ______________________________________                                         Treatment with Soligos as indicated were the same as that in FIG. 7B. At      the end of the experiment (3 weeks), tumor extracts were prepared as          previously described (Ally, S. et al., Cancer Res. 49:5650-5655 (1980))       and immunoblotting and immunoprecipitation of RI.sub.α  and             RII.sub.β, respectively, were performed as previously described by       Ally,S., et al., Proc. Natl. Acad. Sci. USA 85:6319-6322 (1988) and           Tortora, G., et al., Proc. Natl. Acad. Sci. USA 87:705-708 (1990). Data       are from quanitification by densitometric scanning of autoradiograms. Dat     are expressed relative to levels in control tumors (no treatment), which      are set to equal to one as an arbitrary unit.                                 Data represents an average ± S.D. of 7 tumors.                        

In the following sequence listing, Seq ID No: 1 represents an antisensesequence corresponding to the first 7 N-terminal codons for RI.sub.α.Seq ID No: 2 represents an antisense sequence corresponding to the8^(th) -13^(th) codon for RI.sub.α. Seq ID No: 3 represents an antisensesequence corresponding to the 14^(th) -20^(th) codon for RI.sub.α. SeqID No: 4 represents an antisense sequence corresponding to the 94^(th)-100^(th) codon for RI.sub.α. Seq ID No: 5 represents an antisensesequence corresponding to the 1^(st) -100^(th) codon for RI.sub.α. SeqID No: 6 represents the sense sequence corresponding to the 1^(st)-100^(th) codon for RI.sub.α.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCGGTACTGCCAGACTCCAT21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGTGCCTCCTCACTGGC 18                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAGCTCACATTCTCGAAGGCT21                                                       (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATAGCACCTCGTCGCCTCCT21                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 bases                                                          (B) TYPE: Nucleic acid                                                       (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATAGCACCTCGTCGCCTCCTACCTTTAACCACTGGGTTGGGTGGAGGAGGAGAAATCTC60                ATCCTCCCTTGAGTCTGTACGAGTGCCTGC TTTCTGCAGATTGTGAATCTGTTTTGCCTC120              CTCCTTCTCCAACCTCTCAAAGTATTCCCTGAGGAATGCCATGGGACTCTCAGGTCGAGC180               AGTGCACAACTGCACAATAGAATCTTTGAGCAGTGCTTGAATGTTATGCTTCTGGACGTA240               GAGCTCACATTCTCGAAGG CTGCGTGCCTCCTCACTGGCGGCGGTACTGCCAGACTCCAT300              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 bases                                                         (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATGGAGTCTG GCAGTACCGCCGCCAGTGAGGAGGCACGCAGCCTTCGAGAATGTGAGCTC60               TACGTCCAGAAGCATAACATTCAAGCACTGCTCAAAGATTCTATTGTGCAGTTGTGCACT120               GCTCGACCTGAGAGACCCATGGCATTCCTCAGGGAATACTTTGAGAGGTTGGAGAAGGAG18 0              GAGGCAAAACAGATTCAGAATCTGCAGAAAGCAGGCACTCGTACAGACTCAAGGGAGGAT240               GAGATTTCTCCTCCTCCACCCAACCCAGTGGTTAAAGGTAGGAGGCGACGAGGTGCTATC300               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B ) TYPE: Nucleic acid                                                       (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGATCGATCGATCGATCGTAC21                                                   

I claim:
 1. An RNA or DNA antisense oligonucleotide of 15 to 30nucleotides in length and complementary to a region of the first 100N-terminal codons of RI.sub.α (Sequence ID No. 6).
 2. The antisenseoligonucleotide of claim 1 which is DNA.
 3. The antisenseoligonucleotide of claim 1 which is a 21-mer (Sequence ID No. 1).
 4. Theantisense oligonucleotide of claim 1 which is an 18-mer (Sequence ID No.2).
 5. The antisense oligonucleotide of claim 1 which is a 21-mer(Sequence ID No. 3).
 6. The antisense oligonucleotide of claim 1 whichis a 21-mer (Sequence ID No. 4).
 7. The antisense oligonucleotide ofclaim 1 which is DNA and which is complementary to RIα (Sequence ID No.6).
 8. The antisense oligonucleotide of any one of claims 1-6 in whichthe internucleotide linkages are phosphodiesters.
 9. The antisenseoligonucleotide of any one of claims 1-6 in which the internucleotidelinkages are phosphorothioate phosphodiesters.
 10. A pharmaceuticalcomposition comprising at least one antisense oligonucleotide of claim 1and a pharmaceutically acceptable carrier.
 11. The composition of claim10 comprising two antisense oligonucleotides which are complementary toadjacent regions in the first 100 N-terminal codons of RIα (Sequence IDNo. 6), and a pharmaceutically acceptable carrier.
 12. The compositionof claim 10 wherein said pharmaceutically acceptable carrier is asterol.
 13. The composition of claim 10 wherein said pharmaceuticallyacceptable carrier is a liposome.
 14. The composition of claim 10wherein said antisense oligonucleotide is DNA.
 15. The composition ofclaim 10 wherein said antisense oligonucleotide is a 21-mer (Sequence IDNo. 1).
 16. The composition of claim 10 wherein said antisenseoligonucleotide is an 18-mer (Sequence ID No. 2).
 17. The composition ofclaim 10 wherein said antisense oligonucleotide is a 21-mer (Sequence IDNo. 3).
 18. The composition of claim 10 wherein said antisenseoligonucleotide is a 21-mer (Sequence ID No. 4).
 19. The composition ofclaim 10 wherein said antisense oligonucleotide is complementary to RIα(Sequence ID No. 6).
 20. The composition of claim 10 wherein theinternucleotide linkages are phosphodiesters.
 21. The composition ofclaim 10 wherein the internucleotide linkages are phosphorothioatephosphodiesters.